In 1958, Dr Robert Guthrie was approached by Dr Robert Warner seeking aid to develop a better means to measure phenylalanine in the blood of newborns. Of this interaction was born the bacterial inhibition assay and the beginning of population-based newborn screening for phenylketonuria (PKU). Early identification of affected newborns avoided irreversible brain damage using a phenylalanine-restricted diet. While the BIA has been replaced by tandem mass spectrometry as the means to measure phenylalanine, PKU remains the paradigm for a disorder effectively treated by prospective identification of asymptomatic patients. PKU results from defects in phenylalanine hydroxylase (PAH) causing an inability to convert phenylalanine to tyrosine. Treating PKU by dietary means remained largely unchanged until several groups identified a sub- set of patients treatable using 6R-tetrahydrobiopterin (BH4), the obligatory co-factor of the PAH enzyme, without the phenylalanine restricted diet. While BH4-responsive patients are skewed to mild PKU and hyperphenyla'ianemia; classic PKU patients have been characterized as BH4 responsive. Analysis of the PAH gene is becoming an important aspect to determining BH4 response. Comprehensive analysis of the PAH gene may be performed using the universally collected newborn screening dried blood card as a source of DNA. Using the dried blood card and the emerging technology of high-resolution melt profiling, comprehensive analysis of PAH may be easily completed within 1.5 days of abnormal newborn screening results. Genotypic data will be in hand when results of the physiological Phe/ BH4 loading test are complete. Combining physiological analysis and genetic analysis will lead to effective identification of BH4 responsive PKU patients. Cataloging PAH mutations resulting in BH4 responsive disease is underway thus developing a sensitive, rapidly, and cost effective means to analyze the PAH gene will have utility to clinicians and researchers. Herein is proposed the use of high resolution melt profiling to develop a simplified and streamlined means of assessing the coding sequence and intronic regions critical to mRNA processing in the PAH gene. High resolution melt profiling is rapid with sensitivity at least equal to DNA sequence analysis and in excess of other pre-sequence scanning technologies. High resolution melt profiling will play a role to identify BH4 responsive PKU patients.